eDNA PCR test
Geovana Leon
6/11/2019
Degenerated PCR Primer L14912-CYB, H115149-CYB (Fish), amplify ~285 pb, Minamoto et al. 2012
PCR #1 May/15/2019. Pag 19
PCR reaction
- 30 ng/ul DNA aprox
- 1 ul primer (10Mm)
- Biomix 12.5 ul
- 25 ul total reaction
SAMPLES
| Sample | Volume (ul) |
|---|---|
| IPT 1 | 0.7 |
| IPT 2 | 0.5 |
| IPT 3 | 0.5 |
| IPT 4 | 0.5 |
| IPT 5 | 0.5 |
| IPT 6 | 1.2 |
| GSO 1 | 0.7 |
| GSO 2 | 1.2 |
| GSO 3 | 0.5 |
| GSO 4 | 0.7 |
| GSO 5 | 1 |
| GSO 6 | 1 |
| GSO 7 | 1.5 |
| G 2 | 1 |
*1 NEGATIVE
Cycling conditions (Minamoto et al. 2012)
- 2 min to 94°C
- 35 cycles: 94°C 30 s, 50°C 30 s, 72°C 30 s,
- 10 min to 72°C
- 4°C forever
PCR CYB May/15/2019
1% gel, GelGreen, 1:30 hour at 100v
The samples didn´t amplify, the next test I will use Prada´s cycling condition and 40°C annealing temperature.
PCR #2 May/20/2019. Pag 22
PCR reaction
- 30 ng/ul DNA
- 0.5 ul primer (10Mm)
- Biomix 12.5 ul
- 25 ul total reaction
SAMPLES
IPT 1, IPT 2, GSO 1, GSO 2, E 1, E 2, A 1 (1 NEG)
Prada´s Cycling conditions
- 2 min to 94°C
- 90 s 50°C
- 2 min to 72°C
- 36 cycles: 94°C for 45 s, 48°C for 1 min, 72°C for 90 s,
- 1 cycle: 94°C for 40 s, 50°C for 1 min, 72°C for 10 min,
- 12°C forever
I tried: primer 0.5 ul and new cycling condition (Prada). I used samples from Indian Point (IPT), Graduate School of Oceanografphy Beach (GSO) and old extractions.
PCR #3 May/20/2019. Pag 23
PCR reaction
- 30 ng/ul DNA
- 1 ul primer (10Mm)
- Biomix 12.5 ul
- 25 ul total reaction
SAMPLES
E 1, E 2, A 1, D 2, A 3, I 2, G 1 (1 NEG)
I used Prada`s Cycling conditions and just used old extractions
PCR CYB May/20/2019. Pag 22 y 23
1% gel, GelGreen, 1:30 hour at 100v
With primer 0.5 ul. Just old extraction amplified (Pag 22)
With primer 1 ul (Pag 23).
PCR #4 May/29/2019. Pag 31
PCR reaction
- 30 ng/ul DNA aprox
- 1 ul primer (10Mm)
- Biomix 12.5 ul
- 25 ul total reaction
- 2 ul BSA
SAMPLES
| Sample | Extraction |
|---|---|
| D 2 | Old extraction |
| E 1 | Old extraction |
| 1B | GLfilter, ZYMO KIT |
| 3B | GL filter, ZYMO KIT |
| 5B | GL filter, ZYMO KIT |
| 2B | CP filter, ZYMO KIT |
| 4B | CP filter, ZYMO KIT |
| 6B | CP filter, ZYMO KIT |
| 1A | GL filter, QIAGEN KIT |
| 3A | GL filter, QIAGEN KIT |
| 5A | GL filter, QIAGEN KIT |
| 2A | CP filter, QIAGEN KIT |
| 4A | CP filter, QIAGEN KIT |
| 6A | CP filter, QIAGEN KIT |
| IPT 1 (30) | Indian Point extraction |
| IPT 2 (30) | Indian Point extraction |
| IPT 3 (30) | Indian Point extraction |
| GSO 1 (30) | Graduate School of Oceanografphy Beach |
| GSO 2 (30) | Graduate School of Oceanografphy Beach |
| GSO 3 (30) | Graduate School of Oceanografphy Beach |
| GSO 4 (30) | Graduate School of Oceanografphy Beach |
*1 NEGATIVE
I test Prada’s Cycling conditions (50°C), BSA, two differents filters and two differents extraction kits
PCR CYB May/29/2019. Pag 31
1% gel, GelGreen, 1:30 hour at 100v
- Just amplified one new extraction 5A (GL filter, QIAGEN KIT),
- I will try 48°C annealing again, 20 ng/ul DNA template, 1.5 ul BSA.
PCR #5 May/31/2019. Pag 33
PCR reaction
- 20 ng/ul DNA aprox
- 1 ul primer (10Mm)
- 12.5 ul Biomix
- 25 ul total reaction
- 1.5 ul BSA
SAMPLES
| Sample | Extraction |
|---|---|
| E 1 | Old extraction |
| 1B | GLfilter, ZYMO KIT |
| 2B | CP filter, ZYMO KIT |
| 1A | GL filter, QIAGEN KIT |
| 2A | CP filter, QIAGEN KIT |
| IPT 1 (30) | Indian Poin extraction |
| GSO 1 (30) | Graduate School of Oceanografphy Beach |
- 1 NEGATIVE
I test Prada’s Cycling conditions (48°C), 1.5 ul BSA, 20 ng/ul DNA template
PCR CYB May/31/2019. Pag 33
1% gel, GelGreen, 1:30 hour at 100v
- Contamination :(
- I will try another samples and new H20.
PCR #6 Jun/12/2019. Pag 34
PCR reaction
- 20 ng/ul DNA aprox
- 1 ul primer (10Mm)
- 12.5 ul Biomix
- 25 ul total reaction
- 1.5 ul BSA
SAMPLES
| Sample | Extraction |
|---|---|
| E 1 | Old extraction |
| 3B | GLfilter, ZYMO KIT |
| 4B | CP filter, ZYMO KIT |
| 3A | GL filter, QIAGEN KIT |
| 4A | CP filter, QIAGEN KIT |
| IPT 2 (30) | Indian Poin extraction |
| GSO 2 (30) | Graduate School of Oceanografphy Beach |
- 1 NEGATIVE
I test Carlos’ Cycling conditions (48°C), new H20, new samples
1% gel, GelGreen, 1:30 hour at 100v
- Contamination :(
- I will use new reagents.
PCR #7 Jun/12/2019. Pag 35
PCR reaction
- 20 ng/ul DNA aprox
- 1 ul primer (10Mm)
- 12.5 ul Biomix
- 25 ul total reaction
- 1.5 ul BSA
SAMPLES
| Sample | Extraction |
|---|---|
| E 1 | Old extraction |
| 3B | GLfilter, ZYMO KIT |
| 4B | CP filter, ZYMO KIT |
| 3A | GL filter, QIAGEN KIT |
| 4A | CP filter, QIAGEN KIT |
| IPT 2 (30) | Indian Poin extraction |
| GSO 2 (30) | Graduate School of Oceanografphy Beach |
- 1 NEGATIVE
Prada’s Cycling conditions (48°C), new reagents
PCR CYB Jun/13/2019. Pag 34
1% gel, GelGreen, 1:30 hour at 100v